The granule exocytosis model of lymphocyte-mediated cytotoxicity postulates an antigen-triggered rapid secretion of preformed cytotoxic mediators into the synapse-like space between the cytotoxic lymphocyte and its bound target. The generally accepted cytotoxic mediators are perforin and the granzyme subfamily of serine proteases. mRNA encoding a novel member of the thiol protease family, cathepsin W, has been shown to be expressed exclusively in NK and cytotoxic T lymphocytes, suggesting a functional role in cytotoxicity, although a recent paper reported that cathepsin W knockout lymphocytes have normal cytotoxic activity. While nothing is known about cathepsin W protease activity, the protein has been reported by another group to be localized in the endoplasmic reticulum,. We hypothesize that cathepsin W is a granule cytotoxic mediator with functionally redundant protease activity similar to granzymes. In order to study cathepsin W, we expressed human procathepsin W in E. coli and made a series of monoclonal antibodies against it. Western blots of extracts of NK cells and to a lesser extent CTL show mAb reactivity with a single 25-30kD band, where processed, catalytically active cathepsin W is expected. Other mAb react with a 45kD band, consistent with procathepsin W. When human NK92 cells were stimulated with PMA and ionomycin to trigger granule exocytosis, cathepsin W was depleted from the cells and released into the medium within 3 hours and similar results were seen when human CD8+ CTL were treated with anti-CD3. In both cases cat W release paralleled degranulation as measured by granzyme A release. Fluorescence microscopy on NK cells with anti-cathepsin W mAbs show that mature cathepsin W is expressed within the cytoplasm in a vesicular/granular pattern colocalizing with stains for perforin and granzyme A, while procathepsin W is seen in a more diffuse pattern consistent with endoplasmic reticulum. Currently we are using anti-cathepsin W mAb to purify the mature enzyme from NK92 cells and allow study of its enzymatic properties.We have examined the functional status of granule exocytosis in nave, memory, and effector subpopulations of human blood T lymphocytes phenotypically defined by surface markers. When purified directly from blood and immediately assayed by redirected cytotoxicity, only CD8+ effector T cells showed cytotoxic activity. After anti-CD3/anti-CD28 crosslinking, memory T cells became cytotoxic after 48 hours of culture, while both nave and memory cells were cytotoxic after 72 hours. When fixed and permeabilized blood lymphocytes were analyzed by flow cytometry, the effector subset of CD8+ T cells expressed the granule markers granzyme A and B and perforin most strongly. There was limited granule marker expression in CD8+ effector memory cells, while other CD8+ T cell subsets and all CD4+ subsets were negative for all granule markers tested. Both CD4+ and CD8+ blasts were positive for all granule markers, and both cells showed activation-induced surface expression of the lysosomal membrane marker CD107a.